Project summary/abstract Alterations in excitable domains along myelinated axons, the axon initial segment (AIS) and the nodes of Ranvier, are key pathophysiologies in various neurodegenerative conditions. At the paranodes, which flank both sides of the node, myelinating glial cells form junctions with axons to assemble and maintain the nodal protein complex. The cellular and molecular mechanisms of how these domains are disrupted (e.g. AIS shortening, loss of paranodal junctions) in disease conditions, however, remain poorly understood, thereby limiting the field?s ability to manipulate the AIS and nodes for treatment. Our application addresses this critical gap in knowledge of nervous system pathophysiology. Our preliminary data have identified methylglyoxal (MG), a highly reactive byproduct of glucose metabolism, as a potential mediator for AIS and nodal disruption. The overall objective of this application is to identify a critical molecular link in the process of MG-induced AIS and nodal/paranodal disruption. Our preliminary data suggest that calpains, calcium-dependent intracellular cysteine proteases, are involved in this process. The central hypothesis is that methylglyoxal disrupts AIS and nodal/paranodal protein complexes via calpain activation and inhibits nervous system function. We will test this hypothesis via three Specific Aims. Aim 1: Determine the extent of MG-induced AIS shortening and nodal disruption. Using in vitro, ex vivo, and in vivo models, this aim will determine the extent of AIS and node/paranode changes induced by increased MG and the effects on neuronal network activity and nerve conduction along myelinated axons. Aim 2: Determine to what degree calpain inhibition prevents the effects of MG on the AIS and nodes. Using a pharmacological calpain inhibitor, this aim will determine the extent of beneficial effects by calpain inhibition in preventing MG- induced AIS/node changes and neuronal dysfunction. Aim 3: Determine to what degree calpain over-activation exacerbates the effects of MG on the AIS and nodes. Using mutant mice lacking calpastatin, an endogenous calpain inhibitor, this aim will provide genetic confirmation of the role of calpain over-activation in MG-induced AIS/node changes and neuronal dysfunction. This application is conceptually innovative, since completion of the aims will determine the effects of MG on the AIS and the nodes of Ranvier, which have not previously been reported. The proposed research is significant, because it is expected that completion of the aims will provide important new information to develop potential treatments for a wide variety of neurodegenerative conditions associated with disruption of the AIS and nodes. Our results will validate MG and calpains as potential targets for future therapies aimed at treatment of a wide variety of neurodegenerative conditions and ultimately provide a sustained and powerful influence on the field.